Novel phenoxazinone derivatives as enzyme substrates and use thereof as indicator in the detection of microorganisms with peptidase activity

ABSTRACT

The present invention relates to novel enzymatic substrates with the following general formula:  
                 
         where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , A and X are as defined in claim 1, the reaction media comprising the same and the use thereof for the detection and/or identification and/or quantification of microorganisms expressing at least one peptidase activity.

The present invention relates to novel chromogenic enzymatic substratesfor detecting peptidase activity. These substrates can be used inapplications comprising an enzymatic hydrolysis step that produces aphysicochemical signal, in particular in microbiology, biochemistry,immunology, molecular biology, histology, etc. Compared with existingsubstrates, most of which are fluorigenic, the chromogenic substrates ofthe invention can be used in particular in a gelled medium for detectingmicroorganisms since they produce a coloration that does not diffuse inthe reaction medium and is therefore concentrated in the colonies.

The invention also relates to reaction media containing such substrates,to the use of the substrates or of the media for detecting Gram-negativebacteria, Gram-positive bacteria and yeast expressing peptidaseactivity, and to methods of use.

The name aminopeptidase is generally given to an enzyme capable ofcleaving, by hydrolysis, the amide group formed between an acyl of anamino acid and a primary amine, and the name peptidase is given to anenzyme capable of cleaving, by hydrolysis, the amide group formedbetween the acyl residue of a peptide and a primary amine. In thepresent application, the term “peptidase” can denote, as appropriate,both a peptidase and an aminopeptidase as defined above.

Chromogenic enzymatic substrates for detecting peptidase activity thatdo not diffuse are described and already known from the state of theart. Thus, such substrates are covered by patent applicationsWO-A-98/04735 and WO-A-99/38995 filed by the applicant. However, thesesubstrates exhibit various drawbacks: they are difficult to synthesize,the purity is low and the yields are low. In addition, for use inculture media, it is necessary to define a medium composition that isvery precise in order to observe a color. None of the other substratescurrently described can be used in solid media for detectingmicroorganisms in mixed cultures.

Molecules derived from phenoxazinone are known for their ability toproduce fluorescence. They can be used:

as acid-base indicators, as described for example in Stuzka, V. et al.,1963, Collection Czech. Chem. Commun., 28, 1399-1407, or else

-   -   as fluorescent labels, for example for following conformational        modifications of proteins, as described in Nakanishi J. et al.,        2001, Analytical Chemistry, 73(13), 2920-2928.

No phenoxazinone derivative has ever been used as an enzymaticsubstrate.

In accordance with the present invention, novel chromogenic enzymaticsubstrates for detecting microorganisms expressing peptidase activityare proposed. The invention also relates to reaction media containingsuch substrates, and also to the use of the substrates or of the mediafor detecting peptidase activities, and to methods of use.

In fact, the applicant has found, surprisingly, that it is possible todetect microorganisms expressing peptidase activity by using chromogenicphenoxazinone derivatives that produce a coloration that does notdiffuse in the reaction medium, and is therefore concentrated in thecolonies, the peptidase activity being demonstrated by a modification ofthe coloration of the colonies in the culture medium.

After seeding of the reaction media containing the substrates of theinvention with the microorganisms to be tested, colonies that arecolorless to white are observed when the latter are not capable ofhydrolyzing the substrate. On the other hand, colored colonies areobserved when they are capable of hydrolyzing the substrate of theinvention.

The phenoxazinone derivatives of the invention are both chromogenic andfluorigenic and have the advantage of good detection sensitivity.

Thus, a subject of the present invention is chromogenic ezymaticsubstrates of formula (I):

in which

R₁ and R₂ form, with the phenyl ring to which they are attached, anaphthalene ring of formula:

or an optionally substituted coumarin ring of formula:

or else R₁ and R₂ each independently represent a hydrogen atom, a C₁-C₆alkyl group, a halogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an—SO₃H group or a sulfonamide group,

R₃ and R₄ form, with the phenyl ring to which they are attached, anoptionally substituted naphthalene ring of formula:

or else R₃ and R₄ each independently represent a hydrogen atom, ahalogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an —SO₃H group or asulfonamide group, it being understood that:

(i) at least one among R₁/R₂ and R₃/R₄ forms, with the phenyl ring towhich it is attached, an optionally substituted naphthalene or coumarinring as defined above, and

(ii) when R₁ and R₂ form, with the phenyl ring to which they areattached, an optionally substituted coumarin ring, R₃ and R₄ do notform, with the phenyl ring to which they are attached, an optionallysubstituted naphthalene ring,

R₅ and R₆ each independently represent a hydrogen atom, a halogen atom,a —C(O)OR′ group, a C(O)NR′R″ group, or a C₁-C₆ alkyl group,

it being understood that R₆ represents a halogen atom when R₁/R₂ andR₃/R₄ each form, with the phenyl ring to which they are attached, anaphthalene ring,

R₇ and R₈ each independently represent a hydrogen atom, a C₁-C₆ alkylgroup, an aralkyl group, an aryl group, a carboxyalkyl group, a carboxylgroup or a sulfonic acid group,

or else R₇ and R₈, together with the two carbon atoms to which they areattached, form a C₄-C₆ ring,

R₉ represents a hydrogen atom, a bromine atom, a chlorine atom, abenzoyl group, a —CO₂H group or an —SO₃H group,

it being understood that, when R₉ is different from a hydrogen atom,then R₅ is a hydrogen atom,

R′ represents a hydrogen atom or a C₁-C₆ alkyl group,

R″ represents a hydrogen atom or a C₁-C₆ alkyl group,

or else R′ and R″, together with the nitrogen atom to which they areattached, form a heterocyclic ring containing one or more hetero atoms,

A represents at least one amino acid, and

X represents a blocking agent or nothing.

According to the invention, the term “aryl” is in particular intended tomean a C₆-C₁₀ aromatic ring, in particular phenyl, benzyl, 1-naphthyl or2-naphthyl. The same is true for the aryl part of the aralkyl groups.

The alkyls according to the invention, in the aralkyl and carboxyalkylgroups, are also C₁-C₆.

The term “C₁-C₆ alkyl” is intended to mean a straight or branched alkylhaving from 1 to 6 carbon atoms. By way of example, mention may be madeof methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, isopentyland hexyl.

The term “halogen atom” is intended to mean chlorine, bromine, iodineand fluorine.

The term “hetero atom” is intended to mean an atom other than a carbonatom, such as O, N or S.

The heterocyclic rings that R′ and R″ can form may be of any size, butthey preferably contain from 5 to 7 ring members.

Examples of a heterocyclic ring comprise the morpholine, piperazine,piperidine, pyrrolidine and imidazolidine ring.

The various naphthalene and coumarin rings formed by the substituentsR₁/R₂ and R₃/R₄ according to the description of the invention arerepresented by including dashed lines completed with the correspondingsubstituents in the interests of clarity, and make it possible tovisualize the position of said rings in the phenoxazinone derivatives offormula (I) of the invention.

The blocking agents according to the invention comprise any blockingagent known to those skilled in the art which is capable of protectingamines. By way of example, mention may be made of t-butoxycarbonyl(N-tBOC), 9-fluorenyloxycarbonyl, a solubilizing agent such as succinyl,or else a non-metabolizable, i.e. non-natural, amino acid such aspipecolic acid.

The blocking agents are not systematically present in the compounds ofthe invention. In this case, when the compounds of the invention do nothave a blocking agent (X is nothing), the compounds of the invention arein the form of a salt such as chloride, bromide or trifluoroacetate.

The amino acids that are represented by A in formula (I) are any aminoacid known to those skilled in the art.

According to one embodiment of the invention, A represents an amino acidor a peptide having at most 10 amino acids in which the amino acids areidentical or different. Preferably, for reasons of substrate costs, Arepresents an amino acid or a peptide having at most 4 amino acids inwhich the amino acids are identical or different.

According to one embodiment, the compounds of the invention have theformula (I):

in which

R₁ and R₂ form, with the phenyl ring to which they are attached, anaphthalene ring of formula:

or an optionally substituted coumarin ring of formula:

or else R₁ and R₂ each independently represent a hydrogen atom, a C₁-C₆alkyl group, a halogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an—SO₃H group or a sulfonamide group,

R₃ and R₄ form, with the phenyl ring to which they are attached, anoptionally substituted naphthalene ring of formula:

or else R₃ and R₄ each independently represent a hydrogen atom, ahalogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an —SO₃H group or asulfonamide group, it being understood that:

(i) at least one among R₁/R₂ and R₃/R₄ forms, with the phenyl ring towhich it is attached, an optionally substituted naphthalene or coumarinring as defined above, and

(ii) when R₁ and R₂ form, with the phenyl ring to which they areattached, an optionally substituted coumarin ring, R₃ and R₄ do notform, with the phenyl ring to which they are attached, an optionallysubstituted naphthalene ring,

R₅ and R₆ each independently represent a hydrogen atom, a halogen atom,a —C(O)OR′ group, a C(O)NR′R″ group, or a C₁-C₆ alkyl group, it beingunderstood that

(i) R₆ represents a hydrogen atom when R₁ and R₂ form, with the phenylring to which they are attached, a naphthalene or coumarin ring, and

(ii) R₆ represents a halogen atom when R₁/R₂ and R₃/R₄ each form, withthe phenyl ring to which they are attached, a benzene ring,

R₇ and R₈ each independently represent a hydrogen atom, a C₁-C₆ alkylgroup, an aralkyl group, an aryl group, a carboxyalkyl group, a carboxylgroup or a sulfonic acid group,

or else R₇ and R₈, together with the two carbon atoms to which they areattached, form a C₄-C₆ ring,

R₉ represents a hydrogen atom, a bromine atom, a chlorine atom, abenzoyl group, a —CO₂H group or an —SO₃H group,

it being understood that, when R₉ is different from a hydrogen atom,then R₅ is a hydrogen atom,

R′ represents a hydrogen atom or a C₁-C₆ alkyl group,

R″ represents a hydrogen atom or a C₁-C₆ alkyl group,

or else R′ and R″, together with the nitrogen atom to which they areattached, form a heterocyclic ring containing one or more hetero atoms,

A represents at least one amino acid, and

X represents a blocking agent or nothing.

According to another embodiment, the compounds of the invention arechosen from the compounds of formula (I) in which R₁ and R₂ form, withthe phenyl ring to which they are attached, a naphthalene ring, or elseR₁ and R₂ form, with the phenyl ring to which they are attached, acoumarin ring, or else R₃ and R₄ form, with the phenyl ring to whichthey are attached, a naphthalene ring, the other substituents being asdefined above, it being understood that, when R₁/R₂ forms a naphthaleneor coumarin ring with the phenyl ring to which they are attached, R₃/R₄do not at the same time form a naphthalene ring with the phenyl ring towhich they are attached, and vice versa.

Thus, according to this embodiment, the enzymatic substrates arecompounds of formula (I) below:

in which

R₁ and R₂ form, with the phenyl ring to which they are attached, anaphthalene ring of formula:

or an optionally substituted coumarin ring of formula:

or else R₁ and R₂ each independently represent a hydrogen atom, a C₁-C₆alkyl group, a halogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an—SO₃H group or a sulfonamide group,

R₃ and R₄ form, with the phenyl ring to which they are attached, anoptionally substituted naphthalene ring of formula:

or else R₃ and R₄ each independently represent a hydrogen atom, ahalogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an —SO₃H group or asulfonamide group, it being understood that: one and only one amongR₁/R₂ and R₃/R₄ forms, with the phenyl ring to which it is attached, anoptionally substituted naphthalene or coumarin ring as defined above,

R₅ and R₆ each independently represent a hydrogen atom, a halogen atom,a —C(O)OR′ group, a C(O)NR′R″ group, or a C₁-C₆ alkyl group,

R₇ and R₈ each independently represent a hydrogen atom, a C₁-C₆ alkylgroup, an aralkyl group, an aryl group, a carboxyalkyl group, a carboxylgroup or a sulfonic acid group,

or else R₇ and R₈, together with the two carbon atoms to which they areattached, form a C₄-C₆ ring,

R₉ represents a hydrogen atom, a bromine atom, a chlorine atom, abenzoyl group, a —CO₂H group or an —SO₃H group,

it being understood that, when R₉ is different from a hydrogen atom,then R₅ is a hydrogen atom,

R′ represents a hydrogen atom or a C₁-C₆ alkyl group,

R″ represents a hydrogen atom or a C₁-C₆ alkyl group,

or else R′ and R″, together with the nitrogen atom to which they areattached, form a heterocyclic ring containing one or more hetero atoms,

A represents at least one amino acid, and

X represents a blocking agent or nothing.

According to one embodiment of the invention, A represents an amino acidor a peptide having at most 10 amino acids in which the amino acids areidentical or different. Preferably, A represents an amino acid or apeptide having at most 4 amino acids in which the amino acids areidentical or different.

According to a particular embodiment, the compounds of the invention areenzymatic substrates with the following formula (Ia):

in which R₅, R₆ and A and X are as defined above.

The compounds of formula (Ia) are compounds of formula (I) in which theradicals R₁ and R₂ form, with the phenyl ring to which they areattached, a naphthalene ring and R₃ and R₄ are each a hydrogen atom.

Preferably, in the compounds of formula (Ia), R₅ represents a hydrogenatom, R₆ represents a hydrogen atom or a halogen atom, such as achlorine atom, A is an amino acid chosen from leucine, proline andalanine, and X is the t-butoxycarbonyl blocking agent or nothing.

According to another particular embodiment, the compounds of theinvention are enzymatic substrates of formula (Ib):

in which R₅, R₇, R₈, A and X are as defined above.

The compounds of formula (Ib) are compounds of formula (I) in which R₁and R₂ form, with the phenyl ring to which they are attached, a coumarinring and R₃, R₄ and R₆ are each a hydrogen atom.

Preferably, in the compounds of formula (Ib) of the invention, R₅ is ahydrogen atom, R₇ and R₈ each independently represent a hydrogen atom, aC₁-C₆ alkyl group, an aralkyl group, an aryl group or a carboxyalkylgroup, or R₇ and R₈, together with the two carbon atoms to which theyare attached, form a C₄-C₆ ring, A is an amino acid chosen from leucine,proline and alanine, and X is the t-butoxycarbonyl blocking agent ornothing.

According to yet another particular embodiment, the compounds of theinvention are enzymatic substrates of formula (Ic):

in which R₅, R₆, R₉, A and X are as defined above.

The compounds of formula (Ic) are compounds of formula (I) in which theradicals R₃ and R₄ form, with the phenyl ring to which they areattached, a naphthalene ring and R₁ and R₂ are each a hydrogen atom.

Preferably, in the compounds of formula (Ic), the groups R₅, R₆ and R₉each represent a hydrogen atom, A is an amino acid chosen from leucine,proline and alanine, and X is the t-butoxycarbonyl blocking agent ornothing.

According to yet another embodiment, the compounds of the invention areenzymatic substrates of formula (Id):

in which R₅, R₆, A and X are as defined above.

The compounds of formula (Id) are compounds of formula (I) in which theradicals R₁/R₂ and R₃/R₄ each form, with the phenyl ring to which theyare attached, a naphthalene ring.

The compounds of the invention can be prepared according to severalmethods of production depending on the ring that the radicals R₁/R₂ andR₃/R₄ form, whether R₁/R₂ form, with the phenyl ring to which they areattached, a naphthalene ring, or whether R₁/R₂ form, with the phenylring to which they are attached, a coumarin ring, or whether R₃/R₄ form,with the phenyl ring to which they are attached, a naphthalene ring, orwhether R₁/R₂ and R₃/R₄ each form, with the phenyl ring to which theyare attached, a naphthalene ring.

Thus, the compounds of formula (I) in which R₁ and R₂ form, with thephenyl ring to which they are attached, a naphthalene ring, can beprepared according to the procedure represented in scheme 1 below:

According to scheme 1 above, the compounds of formula (I) in which R₁and R₂ form, with the phenyl ring to which they are attached, anaphthalene ring, are prepared by reaction of an appropriate2-amino-5-nitrophenol compound (a) with appropriate halogenated1,4-naphthoquinone (b), which has been heated beforehand to boilingpoint and then cooled to 25° C., so as to form the corresponding9-nitro-benzo[a]phenoxazinone (c). This compound c is subsequentlyreacted with a mixture of copper II acetylacetonate that is reactedbeforehand with sodium borohydride so as to form the compound (d). Thecompound (d) is subsequently reacted with one or more optionallyprotected amino acids (6) in a bath cooled to approximately −12° C., soas to give the compound of formula (I). It should be noted here that, ofcourse, when A is a single amino acid, A′ in the compound (6)corresponds to A of the compound (I), but comprising an additionalhydroxyl group. In other words, when A is a single amino acid, A′ endswith —C(O)OH, while A is linked to —NH— via —C(O)—, losing —OH. When Ais a chain of at least two amino acids, the last amino acid of A′ is asdescribed above, i.e. it comprises, with respect to the last amino acidof A, an additional hydroxyl group.

The compounds of formula (Ia) in which R₁ and R₂ form, with the phenylring to which they are attached, a naphthalene ring, and R₆ is ahydrogen atom, can also be prepared according to the method described inscheme 1a hereinafter.

According to this scheme 1a, the compounds of formula (Ia) are preparedby reaction of an appropriate 3-acetamidophenol compound (1) with anitrite (2), such as a sodium nitrite (X=Na), a potassium nitrite (X=K),etc., at a temperature of −3° C. The nitroso acetamidophenol (3) thusobtained is subsequently reacted with 1,3-dihydroxynaphthalene (4) in asolvent such as butanol, by heating to a temperature of approximately70° C., then adding concentrated sulfuric acid and continuing theheating to 90° C., and then cooling, so as to form the appropriate9-acetamido-benzo[a]phenoxazin-5-one. The latter compound issubsequently hydrolyzed by heating with moderately concentrated sulfuricacid so as to give the 9-amino-benzo[a]phenoxazin-5-one (5). The laststep is equivalent to that of scheme 1.

The compounds of formula (I) in which R₁ and R₂ form, with the phenylring to which they are attached, a coumarin ring, can be preparedaccording to the procedure represented in scheme 2 hereinafter:

According to scheme 2 above, the compounds of formula (I) in which R₁and R₂ form, with the phenyl ring to which they are attached, a coumarinring, but also in which, as indicated in the general definition of thecompounds of formula (I), R₆ is a hydrogen atom, are prepared byoxidizing and chlorinating the appropriate p-phenylenediamine derivative(7) in the presence of the compound (8) so as to obtainN,N′-dichloro-p-benzoquinonediimine (9) according to the method ofWillstaetter and Mayer (1904, Chem. Ber., 37 :1498). The latter compoundis subsequently reacted in an alcoholic solution with5,7-dihydroxycoumarin (10) so as to obtain the appropriate7-amino-1,2-pyronylphenoxazin-3-one (11). The last step is equivalent tothat of scheme 1.

The compounds of formula (Ib), which are compounds of formula (I) inwhich R₁ and R₂ form, with the phenyl ring to which they are attached, acoumarin ring, can of course be prepared with the method above.

The compounds of formula (I) in which R₃ and R₄ form, with the phenylring to which they are attached, a naphthalene ring, can be preparedaccording to the procedure respresented in scheme 3 below:

According to scheme 3 above, the compounds of formula (I) in which R₃and R₄ form, with the phenyl ring to which they are attached, anaphthalene ring, can be prepared by condensation of appropriate2-naphthol (12) with appropriate 4-nitrophenol (13) according to themethod of Fischer & Hepp (reference 36.2, 1807, 1903) so as to give theappropriate naphthophenoxazone (14). The latter compound (14) issubsequently reacted with hydroxylamine hydrochloride (15) according tothe method of Kehrman & Gottrau (reference 38, 2574, 1905) so as toobtain the hydroxyimine and aminoketone forms (compounds 16 and 17,respectively). The last step is equivalent to that of scheme 1.

The compounds of formula (Ic), which are compounds of formula (I) inwhich R₃ and R₄ form, with the phenyl ring to which they are attached, anaphthalene ring, can of course be prepared with the method above.

Finally, the compounds of the invention of formula (I) in which theradicals R₁/R₂ and R₃/R₄ each form, with the phenyl ring to which theyare attached, a naphthalene ring (compounds of formula (Id)), can beprepared according to the procedure represented in scheme 4 below:

According to scheme 4 above, the compounds of formula (I) in which theradicals R₁/R₂ and R₃/R₄ each form, with the phenyl ring to which theyare attached, a naphthalene ring, can be prepared by condensation ofappropriate 1-amino-2-naphthol-4-sulfonic acid (18) with appropriate2-hydroxy-1,4-naphthoquinone (19) so as to produce the appropriatedinaphthoxazonesulfonic acid (20). This compound (20) is subsequentlyheated in the presence of ammonium so as to produce theaminodinaphthoxazone (21). The last step of this procedure is equivalentto that of scheme 1.

In the above procedures, the starting reactants (compounds (1), (2),(4), (6), (7), (8), (10), (12), (13), (15), (18) and (19)) arecommercially available, in particular from Aldrich.

A subject of the invention is also a reaction medium that uses at leastone chromogenic enzymatic substrate of formula (1) as defined above,alone or in combination with at least one other enzymatic substratespecific for an enzymatic activity different from that detected by thesubstrate according to the invention.

In fact, when microorganisms expressing peptidase activity are seededinto or onto a reaction medium containing the compounds of theinvention, a coloration occurs that does not diffuse in or on thereaction medium, and is therefore concentrated in the colonies.

The term “reaction medium according to the invention” is intended tomean a medium that allows the development of at least one enzymaticactivity of at least one microorganism.

This reaction medium can either serve only as visualizing medium, or asculture medium and visualizing medium. In the first case, themicroorganisms are cultured before seeding and, in the second case, thereaction medium also constitutes the culture medium, which constitutes aparticular embodiment of the invention.

The reaction medium may be solid, semi-solid or liquid. The term “solidmedium” is intended to mean, for example, a gelled medium.

Agar is the traditional solid medium in microbiology for culturingmicroorganisms, but it is possible to use gelatin or agarose. A certainnumber of preparations are commercially available, such as, for example,Columbia agar, Trypcase soy agar, Mac Conkey agar, Sabouraud agar or,more generally, those described in the Handbook of Microbiological Media(CRC Press).

Preferably, when the reaction medium is also a culture medium, it is ingelled form.

The amount of agar in the reaction medium is from 2 to 40 g/l andpreferably from 9 to 25 g/l.

The enzymatic substrates of the invention can be used within a broad pHrange, in particular between pH 5.5 and 10.

The concentration of enzymatic substrate of the invention in thereaction medium is between 0.025 and 0.40 g/l, and it is advantageously0.05 g/l. This is because, at this substrate concentration, bettercoloration contrast is obtained.

The reaction medium may comprise at least one other substrate specificfor an enzymatic activity different from that detected by the substrateaccording to the invention. The enzymatic hydrolysis of the othersubstrate(s) generates a detectable signal that is different from thesignal detected by the substrate of the invention, such as, for example,different colored or fluorescent products, so as to allow thedemonstration such as the detection and/or the identification and/or thequantification of one or more microorganisms.

As other specific substrate, mention may be made of substrates ofindoxyl type, such as 5-bromo-4-chloro-3-indoxyl-β-D-glucoside(Biosynth) or 5-bromo-6-chloro-3-indoxyl-β-D-galactoside (Biosynth), orany other substrate used in the detection of microorganisms.

The concentration of the other specific enzymatic substrate is generallybetween 0.01 and 2 g/l. Those skilled in the art will be able to readilydetermine such a concentration according to the substrate used.

The reaction medium can also comprise one or more elements incombination, such as amino acids, peptones, carbohydrates, nucleotides,minerals, vitamins, antibiotics, surfactants, buffers, phosphate salts,ammonium salts, sodium salts or metal salts. Examples of media aredescribed in the applicant's patent applications EP 656 421 and WO 99/09207.

The enzymatic substrates and reaction media of the invention aretherefore useful in the diagnosis of microorganisms with peptidaseactivity.

Thus, a subject of the present invention is also the use of achromogenic enzymatic substrate of formula (I), or of a reaction mediumas defined above, for the detection and/or identification and/orquantification of microorganisms expressing at least one peptidaseactivity.

The invention also relates to a method for the detection and/oridentification and/or quantification of microorganisms expressing atleast one peptidase activity, characterized in that in consists in:

-   -   providing a reaction medium, as defined above,    -   seeding the medium with a biological sample to be tested,    -   leaving to incubate, and    -   revealing the presence of at least one peptidase activity alone        or in combination with at least one other enzymatic activity        different from this same peptidase activity.

The seeding and incubation steps are widely known to those skilled inthe art.

For example, the incubation temperature is 37° C. As regards theincubation atmosphere, it can equally be anaerobic or aerobic. However,the incubation is preferably carried out under aerobic conditions sincethis improves the enzymatic activity.

The revelation is carried out with the naked eye by visualizing a changein coloration that does not diffuse in the reaction medium, and istherefore concentrated in the colonies.

By way of microorganisms which may be diagnosed by means of theenzymatic substrate of the invention, mention may be made ofGram-negative bacteria, Gram-positive bacteria and yeasts.

By way of Gram-negative bacteria, mention may be made of bacteria of thefollowing genera: Pseudomonas, Escherichia, Salmonella, Shigella,Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus,Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria,Burkholderia, Citrobacter, Hafnia, Edwardsiella and Legionella.

By way of Gram-positive bacteria, mention may be made of bacteria of thefollowing genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus,Listeria, Clostridium, Mycobacteria and Corynebacteria.

Examples of yeasts comprise yeasts of the following genera: Candida,Cryptococcus, Saccharomyces and Trichosporon.

The biological samples to be analyzed are any clinical sample, such as asaliva, blood, urine or stool sample, or any other sample the analysisof which may aid a clinician in putting forward a diagnosis. The samplemay also be a sample of a product derived from, or a base product of,the food and/or pharmaceutical industry, in which it is necessary toeither guarantee the absence of pathogenic microorganisms, or to count acontaminating flora, or to detect specific microorganisms.

The chromogenic substrates of the invention, in which A is alanine, havethe advantage that they make it possible to differentiate Gram-negativebacteria from Gram-positive bacteria.

Thus, another subject of the invention consists of a method fordifferentiating, among bacteria, between those belonging toGram-positive microbes and those belonging to Gram-negative microbes,characterized in that consists in:

-   -   providing a reaction medium, as defined above and in which the        substituent A of the chromogenic substrate is alanine,    -   seeding the medium with a biological sample to be tested,    -   leaving to incubate, and    -   revealing the presence of at least one coloration synonymous        with the presence of a Gram-negative microbe or Gram-negative        microbes.

As regards the chromogenic substrates in which A is proline, they havethe advantage that they make it possible to differentiate the yeast ofthe species Candida albicans from those of the species Candidatropicalis and Candida glabrata.

Thus, another subject of the invention relates to a method fordifferentiating the yeast of the species Candida albicans from those ofthe species Candida tropicalis and Candida glabrata, characterized inthat it consists in:

-   -   providing a reaction medium, as defined above and in which the        substituent A of the chromogenic substrate is proline,    -   seeding the medium with a biological sample to be tested,    -   leaving to incubate, and revealing the presence of at least one        coloration synonymous with the presence of the yeast of the        species Candida albicans.

The invention will be understood more clearly from the followingexamples given by way of nonlimiting illustration.

EXAMPLE 1 Synthesis of 9-aminobenzo[a]phenoxazin-5-one

(Compound (5) with R₃=R₄=R₅=H)

1.1 Preparation of 2-nitroso-5-acetamidophenol

9 g of 3-acetamidophenol (Aldrich) were dissolved in an aqueous solution(100 ml) containing 2.8 g of sodium hydroxide. The solution was cooledto −3° C. using an ice-salt bath and 5 g of sodium nitrite in water (12ml) were added.

A solution of phosphoric acid diluted with an equal amount of water (25ml) was added, using a separating funnel, to the stirred solution. Suchan addition was carried out at a rate such that the temperature wasmaintained at 0° C. or below this temperature. A red-brown precipitatethen formed. After further vigorous stirring for one hour, the pH wasdetermined so as to guarantee complete acidity (pH<2).

The thick suspension thus obtained was filtered and the residue wascarefully washed with cold water so as to remove the excess acid andsalts. After appropriate aspiration, the residue was dried in a vacuumdesiccator. 7.36 g of 2-nitroso-5-acetamidophenol were obtained with ayield of 68.6%.

1.2 Preparation of 9-acetamidobenzo[a]phenoxazin-5-one

1.8 g of the crude product obtained in point 1.1 above and 1.60 g of1,3-dihydroxynaphthalene (Aldrich) were dissolved in 50 ml of butan-1-olwhile at the same time stirring and heating to 70° C. 1 g ofconcentrated sulfuric acid was added dropwise to the heated solution andthe heating was continued to 90° C. After 30 min, the mixture wasallowed to cool. The solid phase was removed by suction filtration andwashing was performed with a little ethanol. After drying, the titlecompound was obtained with a yield of 76%.

The product obtained was subjected to thin layer chromatography onsilica gel plates using ethyl acetate/toluene (3:1) as mobile phase. Alight orange-yellow spot was obtained (R_(f)=0.8).

1.3 Preparation of 9-aminobenzo[a]phenoxazin-5-one

1.5 g of the compound obtained in point 1.2 above were dissolved in aminimal volume of sulfuric acid and water (1:1), while at the same timestirring and heating to 100° C. until a sample taken and diluted inwater, and then extracted in ethyl acetate, no longer showed anystarting product by thin layer chromatography. The dark solution thusobtained was stirred and was heated to boiling point for severalminutes, and was then cooled and poured into an ice-water bath (300 ml).The precipitated base was finely divided, heating was then carried outto 40° C. and the product was left to stand overnight. The supernatantliquid was allowed to separate by settling out, and the suspension ofproduct was filtered and washed with water. After drying, the intendedproduct was obtained with a yield of 78%.

EXAMPLE 2 Aminoacylation of 9-aminobenzo[a]phenoxazin-5-one

(Compound of Formula (I) in which R₁/R₂ form a Naphthalene,R₃=R₄=R₅=R₆=H, A=an amino acid and X=N-t-BOC)

0.52 g (2 mmol) of the amino compound concerned, obtained in Example 1,was dissolved in 15 ml of dimethylformamide (high performance liquidchromatography quality) while at the same time heating, and then in 10ml of tetrahydrofuran. This solution was hydrogenated in a three-neckedflask using hydrogen (produced from sodium borohydrate/acetic acid), andalso 0.1 g of 10% palladium-on-charcoal as catalyst. The dark violetcolor was replaced with a fluorescent green appearance. Thehydrogenation was continued for 30 min, the flask was closed and wasleft to stand overnight.

3 mmol of amino acid protected with N-tBOC were dissolved in 10 ml ofanhydrous THF and the product was cooled to −12° C. (ice/salt bath).0.33 g (3.3 mmol) of N-methylmorpholine was added to the solution thuscooled, and then 0.42 g (3.1 mmol) of isobutyl chloroformate was addedgently at between −12° C. and −9° C. After 5 min, the mixed anhydridereaction mixture above was poured into the stirred solution of reducedamine, the mixture was precooled to at least −5° C., while hydrogen wasintroduced so as to prevent reoxidation. After 10 min, the flask wasclosed and the content was stirred for a further 5 hours at ambienttemperature.

The reaction mixture was filtered and the solvent (THF) was removed byrotary evaporation. The DMF solution was poured into a thoroughlystirred water-ice mixture and the precipitate was filtered off, washedwith water and air-dried. The crude product was dissolved indichloromethane (DCM) and was washed with dilute (0.2M) sodium hydroxideand then with water. After drying with magnesium sulfate, the solventwas eliminated.

In certain cases, the filtration of the DCM extract through a cone ofsilica gel removed the traces of the base material liable to be observedby thin layer chromatography.

The compound obtained in this example can then be deprotected in thefollowing way: the product is dissolved in a small volume of ethylacetate and stirred with an equal amount of ethyl acetate saturated withhydrogen chloride for 1 hour. An excess of anhydrous ether is added andthe precipitated hydrochloride salt is rapidly filtered off, then washedwith additional ether or ether/mineral essence, and then dried in vacuo.

EXAMPLE 3 Synthesis of 9-amino-6-carbethoxybenzo[a]phenoxazin-5-one

(Compound of Formula I in which R₁/R₂ form a Naphthalene and R₃=R₄=R₅=Hand R₆=—C(O)OR′ with R′=C₂H₅)

3.1 Preparation of ethyl 1,3-dihydroxynaphthoate

This compound was produced from diethyl malonate and phenylacetylchloride according to the method of Meyer and Bloch (Org. Synth. Coll.,Vol 3, p 132).

3.2 Preparation of 9-acetamido-6-carbethoxybenzo[a]phenoxazin-5-one

1.8 g (10 mmol) of 2-nitroso-5-acetamidophenol and 2.08 g (9 mmol) ofethyl 1,3-dihydroxynaphthoate were dissolved in 60 ml of butanol whileheating and the mixture was stirred at 70° C. Concentrated sulfuric acidwas gradually added dropwise and the solution was heated gradually toapproximately 90° C. After 30 min, the reaction mixture was cooled andwas kept at 5° C. overnight.

The red product was removed by suction filtration and washed with alittle ethanol.

After drying, the 9-acetamido-6-carbethoxybenzo[a]phenoxazin-5-one wasobtained with a yield of 65%.

3.3 Preparation of 9-amino-6-carbethoxybenzo[a]phenoxazin-5-one

The 9-acetamido-6-carbethoxybenzo[a]phenoxazin-5-one obtained in point3.2 was dissolved in a mixture of sulfuric acid (3 ml) and ethanol (3ml). The mixture was heated to 80° C. while at the same time graduallyadding water (1 ml). The purple color characteristic of the aminerapidly appeared. The hydrolysis was continued until the sample, dilutedwith water, and then extracted in ethyl acetate, no longer showed any9-acetamido-6-carbethoxybenzo[a]phenoxazin-5-one by thin layerchromatography.

The reaction mixture was poured into 150 ml of ice-cold water and theproduct was collected by suction filtration, and then washed with waterand dried. The product was obtained with a yield of 85%.

EXAMPLE 4 Aminoacylation of 9-amino-6-carbethoxybenzo[a]phenoxazin-5-one

The procedure was carried out as described in Example 2, using theproduct obtained in Example 3 and using the appropriate amino acid,protected with N-t-Boc.

For the deprotection of the aminated compound, it was dissolved in 2 mlof trifluoroacetic acid, which operation was followed by precipitationfrom ether. The product was thus obtained in the form of an homogeneousorange powder by thin layer chromatography.

EXAMPLE 5 Synthesis of 9-amino-6-chlorobenzo[a]phenoxazin-5-one

(Compound of Formula I in which R₁/R₂ form a Naphthalene and R₃=R₄=R₅=Hand R₆=Cl)

5.1 Preparation of 9-nitro-6-chlorobenzo[a]phenoxazin-5-one

1.54 g (10 mmol) of 95% pure 2-aminonitrophenol (Aldrich) were added toa suspension of 2.26 g (10 mmol) of 2,3-dichloro-1,4-naphthoquinone(Fluka) in ethanol, heated beforehand to boiling point, and then cooledto 25° C. The mixture was stirred and 1 g of anhydrous sodium acetatewas added. After several hours, an orangey-brown precipitate formed.After continuous stirring for 24 h, the solid was isolated by suctionfiltration, dried and recrystallized from hot acetic acid (yield of65%).

5.2 Preparation of 9-amino-6-chlorobenzo[a]phenoxazin-5-one

130 mg (1 mmol) of copper II acetylacetonate, suspended in 10 ml ofethanol, were stirred with 0.18 g (5 mmol) of sodium borohydride atambient temperature until the formation of a brown compound derived fromthis catalysis (approximately 10 min). 1.29 g (4 mmol) of the compoundobtained in point 5.1 above, in the form of a suspension in 10 ml ofpropan-1-ol, were added to this mixture, followed by 0.37 g (10 mmol) ofsodium borohydride. The mixture was stirred for 3 h at 30° C.

After cooling, the reaction mixture was poured into an ice/watermixture, and the crude product was then recovered by filtration and wasdried. It was purified by dissolving in hot butan-1-ol and by filtrationin order to remove the products containing copper. After concentration,the title compound was crystallized so as to give 0.68 g of product.

EXAMPLE 6 Aminoacylation of 9-amino-6-chlorobenzo[a]phenoxazin-5-one

The procedure was carried out as described in Example 2, using theproduct obtained in Example 5 and using the appropriate amino acid,protected with N-t-Boc.

For the deprotection of the aminated compound, the procedure was alsocarried out as described in Example 2.

EXAMPLE 7 Synthesis of 5-aminobenzo[a]phenoxazin-9-one

(Compound of Formula I in which R₃/R₄ form a Naphthalene andR₁=R₂=R₅=R₆=R₉=H)

7.1 Preparation of Naphthophenoxazone

The method of Fisher & Hepp (above) was used without significantmodification, carrying out the condensation of 4-nitrosophenol and of2-naphthol in glacial acetic acid using zinc chloride as condensationagent.

The crude product was recrystallized from hot toluene/mineral essencewith a yield of 25%.

The 4-nitrosophenol used here is a commercial product obtained fromFluka that was converted in the following way: this product was purifiedby dissolving it in ether, by filtering it through a Phase-Sep paper andby stirring with Norite for one hour. After filtration, the etherealsolution was rotary-evaporated so as to obtain a small volume and cooledso as to obtain a crystalline product of pure nitrosophenol.

7.2 Preparation of the Amine Product

According to the method of Kehrmann & Gottrau (above), 3.0 g of thenaphthophenoxazone obtained in point 7.1 above and 3.0 g ofhydroxylamine hydrochloride were mixed with 200 ml of absolute ethanoland the mixture was gradually heated to boiling point. The red color ofthe naphthophenoxazone was gradually replaced with an orange color andprecipitation of the hydrochloride of the base occurred. The precipitatewas removed by filtration, washed with a little ethanol, and then driedso as to obtain the hydrochloride with a yield of 63%.

1 g of the salt thus obtained was decomposed by heating with water, andthen by cooling so as to give a green residue of the free base. Theproduct was filtered and dissolved in a few ml of ethanol at 40° C.while at the same time adding a sufficient amount of HCl for solution.The fluorescent violet-red solution was heated with a sufficient amountof anhydrous sodium acetate to give the free base (dark green metalneedles). The product was filtered, washed with hot water, and dried toattain a yield of 97%.

EXAMPLE 8 Synthesis of7-amino-1,2-(3′,4′-cyclopenteno-2′-pyronyl)-phenoxazin-3-one

(Compound of Formula I in which R₁/R₂ form a Coumarin, R₇ and R₈,Together with the Two Carbon Atoms to which They are Attached, Form aCyclopentene and R₃=R₄=R₅=R₆=H)

8.1 Preparation of 5,7-dihydroxy-3,4-cyclopentenocoumarin

3.02 g (24 mmol) of phloroglucinol and 3.12 g (20 mmol) of ethyl2-oxocyclopentanecarboxylate were mixed together in a small flask usinga little ethanol. After cooling, 30 ml of a mixture of sulfuricacid/water at 75% mass by mass were added to the stirred mixture. Thestirring was continued at ambient temperature for 48 h. The semi-solidproduct was poured into a thoroughly stirred ice/water mixture andfiltration was carried out. The residue was thoroughly washed withwater, drained by suction and air-dried.

Recrystallization from ethanol gave a product that was homogeneous bythin layer chromatography. The yield was 2.8 g.

8.2 Preparation of the Title Compound

2.18 g (10 mmol) of the 5,7-dihydroxy-3,4-cyclopentenocoumarin obtainedin point 8.1 above were dissolved in 40 ml of hot ethanol and 1.74 g (10mmol) of 1,4-dichloro-p-benzoquinonediimine were added to the stirredsolution. The reaction mixture was slowly brought to reflux above awaterbath for several minutes, during which time the liquid became deepviolet in color. After refluxing for a further 20 min, the reactionmixture was poured into 250 ml of an ice/water mixture containing 2 g ofacetate. The colorant separated in the form of a dark blue precipitate.The precipitate thus obtained was removed by suction filtration and waswashed with water. The dried product (1.5 g) could be recrystallizedfrom acetic acid and butan-1-ol.

EXAMPLE 9 Synthesis of7-amino-1,2-(4′-methyl-2′-pyronyl)phenoxazin-3-one

(Compound of Formula I in which R₁/R₂ form a Coumarin, R₈ is a Methyland R₇=R₃=R₄=R₅=R₆=H)

9.1 Preparation of 5,7-dihydroxy-4-methylcoumarin

A mixture of 2.77 g (22 mmol) of phloroglucinol and 2.6 g (20 mmol) ofethyl acetoacetate were melted together, the mixture was cooled, and 40ml of a mixture of sulfuric acid/water (75% w/w) was added rapidly tothe semi-solid mass. The mixture was stirred for 24 h, during which timea semi-solid mass formed. It was poured into a mixture of ice/water (300ml) containing 5 g of sodium acetate and the precipitate was recoveredby suction filtration.

After repeated washing with water and drying, the crude product wasrecrystallized from hot ethanol so as to give 3.05 g of coumarin.

9.2 Preparation of 7-amino-1,2-(4′-methyl-2′-pyronyl)phenoxazin-3-one

1.92 g (10 mmol) of 5,7-dihydroxy-4-methylcoumarin were dissolved in 50ml of anhydrous methanol. 7.5 g (0.125 mol) of urea were added to thestirred hot solution in order to moderate the exothermicity and toreduce the chlorination of the product. 1.74 g (10 mmol) of1,4-dichlorobenzoquinonediimine were added to the reaction mixture,which had been refluxed for 2 h with stirring.

The dark purple solution was poured into a thoroughly stirred mixture ofwater/ice containing 10 g of sodium acetate. The dark purple precipitatewas removed by vacuum filtration and drying was carried out. The crudeproduct was purified by suspension in 200 ml of methanol heated to 50°C. while at the same time gradually adding to the thoroughly stirredmixture a solution of 5 g of sodium dithionite and 5 g of sodiumcarbonate in 20 ml of water. A yellowy-brown-colored precipitate formed.

The reaction mixture was rapidly filtered so as to isolate a precipitateof the colorant in the dihydro form. It was washed with a mixture ofice/water containing a little sodium dithionite and the precipitate thusmade moist was transferred into 100 ml of methanol. The rapidly stirredsolution was heated to 40° C. and water was gradually added, therebymaking it possible to reoxidize the colorless colorant (in dihydro form)and to obtain a dark purplish-brown precipitate. Finally, 100 ml ofwater were added so as to end the oxidation and the precipitation of thecolorant, which was dried after having been removed by filtration. Thinlayer chromatography indicated only a single purple compound and a fewtraces of the same compound in the dihydro form.

EXAMPLE 10 Synthesis of7-amino-1,2-(3′-carboxyethyl-4′-methyl-2′-pyronyl)phenoxazin-3-one

(Compound of Formula I in which R₁/R₂ form a Coumarin, R₇=Carboxyethyl,R₈=Methyl and R₃=R₄=R₅=R₆=H)

10.1 Preparation of ethyl 5,7-dihydroxy-4-methylcoumarin-3-propanoate

2.77 g (22 mmol) of phloroglucinol and 4.60 g (20 mmol) of diethylacetylglutarate were mixed together, cooled, and stirred with 35 ml of amixture of sulfuric acid/water at 75% mass/mass. The stirring wascontinued for 48 h. The product was isolated by pouring it into astirred ice/water mixture and suction filtration was carried out. Theresidue was washed thoroughly with water and air-dried.

The product was converted directly to the free acid by suspending it inethanol and stirring with an aqueous solution of potassium hydroxide (3molar equivalents). After 4 h, the acid precipitated through theaddition of 2M hydrochloric acid at pH 2. The abundant precipitate wasremoved by suction filtration, it was washed with water and it wasdrained off by suction. After air-drying, an amount of 3.8 g of theintended product was obtained.

10.2 Preparation of the Title Compound

This compound was prepared as in Example 8.1 using1,4-dichloro-p-benzoquinonediimine and the acid obtained in point 10.1above (equimolar amounts). The reaction mixture was poured into anice/water mixture, adding hydrochloric acid until a pH of 2 was obtainedin order to ensure complete precipitation of the free carboxylic acidform of the colorant. The amount of dried product obtained was 2.2 g.

EXAMPLE 11 Detection of Microorganisms Expressing Leucine-PeptidaseActivity 11.1 Preparation of the Detection Media

The detection medium was prepared by mixing 1.4 g of biogelytone(bioMérieux), 0.84 g of meat extract (bioMérieux), 2.24 g of NaCl(Merck), 280 μl of a solution of IPTG(isopropylthio-p-D-galactopyranoside, BIOSYNTH) in water (concentration10 g/l) and 4.2 g of European agar (bioMérieux).

The substrate of the invention obtained in Example 2, in which the aminoacid is leucine (leucine-9-aminobenzo[a]phenoxazin-5-one or Leu-ABP), orelse a substrate of the prior art, which is leucine-aminomethylcoumarin(Leu-AMC, BACHEM), was then added in the following way: 280 ml ofosmosed water were added to the medium, and the mixture was melted in awaterbath at 100° C. The mixture was autoclaved for 15 min at 121° C.and cooled to 50° C. in a waterbath.

The substrate solubilized in DMSO (Merck) was then added according tothe concentrations indicated in Table 1 below: TABLE 1 Medium MediumMedium Medium Medium Medium Medium 1 2 3 4 5 6 7 Leu- 0 mg/l 25 mg/l 50mg/l 100 200 400 ABP mg/l mg/l mg/l Leu- 50 mg/l AMC

The medium was then poured into Petri dishes.

11.2 Seeding of the Microorganism Strains

Ten microorganism strains derived from the Applicant's collection,suspended in physiological saline, were seeded in colonies on each ofthe media. The dishes were incubated at 37° C. for 48 hours. Thecolonies formed were examined visually after incubation for 24 and 48hours. The coloration of these colonies, the diffusion, and also theintensity of this coloration were noted.

11.3 Results

The results were expressed as intensity of coloration using an arbitraryscale ranging from 0 to 4 as a basis and also as diffusion also using anarbitrary scale ranging from 0 to 4 as a basis. These results are givenin Table 2 below, where

T^(a) corresponds to the incubation time, C^(b) corresponds to thegrowth diameter in mm, I^(c) corresponds to the intensity of coloration,Co^(d) corresponds to the color of the colony and D^(e) corresponds tothe diffusion,

B corresponds to fluorescent blue and R corresponds to pink. TABLE 2Medium 1 Medium 2 Medium 3 Medium 4 Activity Activity Activity ActivityStrain T^(a) C^(b) I^(c) Co^(d) D^(e) C^(b) I^(c) Co^(d) D^(e) C^(b)I^(c) Co^(d) D^(e) C^(b) I^(c) Co^(d) D^(e) Escherichia 24 h 1.8 3 B 41.8 1.8 0.0 R 1.7 0.5 R 0.5 coli 48 h 1.8 3 B 4 1.8 1.8 0.25 R 0.25 1.80.5 R 0.5 Citrobacter 24 h 0.8 3 B 4 0.8 0.7 1.5 R 0.5 0.7 1.5 R 0.5freundii 48 h 1 3 B 4 0.8 0.7 2.5 R 0.5 0.7 2.5 R 0.75 Klebsellia 24 h1.8 3 B 4 1.8 1.8 1.5 R 0.25 1.7 1.5 R 0.5 pneumoniae 48 h 1.8 3 B 4 1.81.8 2 R 0.25 1.7 2.5 R 0.75 Enterobacter 24 h 0.8 3 B 4 0.7 0.7 1.5 R0.5 0.5 2.5 R 0.5 cloaccae 48 h 0.8 3 B 4 0.7 0.7 2.5 R 0.5 0.5 3 R 0.75Serratia 24 h 0.5 2 B 4 0.5 0.5 0.5 R 0.4 1 R marcescens 48 h 0.7 3 B 40.7 0.7 2.5 R 0.5 0.5 3 R 0.5 Pseudomonas 24 h 2 1 B 4 2 2 2 0.75 Raeruginosa 48 h 2 3 B 4 2 2 1 R 0.5 2 2.5 R 1 Staphylococcus 24 h 1.72.5 B 4 1.7 0.3 0.2 aureus 48 h 1.7 3 B 4 1.7 0.7 0.25 R 0.25 0.2Enterococcus 24 h 0.4 0.5 B 4 0.4 0.3 0.0 R 0.25 0.3 0.0 R 0.25 faecalis48 h 0.4 1.5 B 4 0.4 0.3 0.0 R 0.25 0.3 0.0 R 0.25 Candida 24 h 0.4 0.25B 4 0.4 0.3 albicans 48 h 0.5 1.5 B 4 0.7 0.5 0.25 R 0.3 Medium 5 Medium6 Medium 7 Activity Activity Activity Strain C^(b) I^(c) Co^(d) D^(e)C^(b) I^(c) Co^(d) D^(e) C^(b) I^(c) Co^(d) D^(e) Escherichia 1.8 0.0 R1.7 1.8 0.75 R coli 1.8 R 1.7 1.8 Citrobacter 0.7 0.75 R 0.8 1 R 0.7 2 Rfreundii 0.7 1.5 R 0.25 0.8 2 R 0.7 2 R Klebsellia 1.7 1.5 R 1.7 2 R 1.72.5 R pneumoniae 1.7 1.5 R 0.0 1.7 1 R 1.8 1 R Enterobacter 1 3 R 1.5 3R 1.5 3 R cloaccae 1.3 3 R 0.25 1.5 3 R 1.7 3 R Serratia 0.4 0.25 R 0.40.4 marcescens 0.5 1.5 R 0.0 0.5 1 R 0.5 0.5 R Pseudomonas 2 0.75 R 20.5 R 2 0.5 R aeruginosa 2 2.5 R 0.5 2 2 R 0.3 2 0.5 R Staphylococcusaureus Enterococcus 0.3 0.3 0.3 faecalis 0.3 0.0 R 0.0 0.3 0.3 0.5 RCandida albicans

The results indicated in Tables 1 and 2 above demonstrate that thechromogenic enzymatic substrates of the invention clearly make itpossible to diagnose microorganisms expressing leucine-peptidaseactivity and that, compared with a substrate of the prior art, a verysmall amount of diffusion around the colony is observed.

EXAMPLE 12 Detection of Microorganisms Expressing Prolyl-Peptidase 12.1Preparation of the Detection Media

The detection media were prepared by mixing 1.68 g of yeast extract(bioMérieux), 1.4 g of biocase (bioMérieux), 1.26 g of malt extract(bioMérieux), 0.08 g of glucose (Merck) and 3.92 g of agar (bioMérieux).

280 ml of osmosed water were added to the powder, and the mixture wasthen melted in a waterbath at 100° C. The product was separated into twoflasks, each of 140 ml. Autoclaving was carried out at 121° C. for 15min and the product was cooled to 50° C. in a waterbath.

L-Prolyl-7-amino-4-methylcoumarin (Pro-AMC, Bachem) was then added tothe first flask as a control andL-prolyl-9-aminobenzo[a]phenoxazin-5-one (Pro-ABP, substrate of theinvention obtained in Example 2, in which the amino acid is L-Proline)was then added to the second flask at the final concentration of 50mg/l.

12.2 Seeding of the Microorganism Strains

Nine microorganism strains derived from the Applicant's collection,suspended in physiological saline, were seeded in colonies on each ofthe media. The dishes were incubated at 37° C. for 48 hours. Thecolonies formed were examined visually after incubation for 24 and 48hours. The size of the colonies, their coloration and also the intensityof this coloration were noted.

12.3 Results

The results are expressed in Table 3 hereinafter, according to the rulesand the nomenclature stated in paragraph 11.3. TABLE 3 Prolyl-peptidaseactivity of microorganisms with the substrates Pro-AMC of the prior artand Pro-ABP of the invention Pro-AMC Pro-ABP T^(a) C^(b) I^(c) Co^(d)C^(b) I^(c) Co^(d) Escherichia coli 24 3 0.5 B 3 0.25 R 48 3 0.5 B 3 3 RKlebsiella pneumoniae 24 2.5 0.25 B 2 48 3 0.25 B 3 Enterococcusfaecalis 24 0.8 0.7 48 1.2 0.7 Candida albicans 24 1.2 2 B 0.8 2 R 482.5 3 B 2.5 2 R Candida guilliermondii 24 0.4 0.3 48 1.8 3 B 1.8 2 RCandida glabrata 24 0.3 0.4 48 1.7 1.8 Candida tropicalis 24 1.2 1.2 482 1.8 Trichosporon beigelii 24 0.7 0.25 B 0.5 48 3 2 B 1.8 2 RSaccharomyces 24 0.3 0.5 cerevisiae 48 1.7 2

The results indicated in the table above demonstrate that thechromogenic enzymatic substrates of the invention clearly make itpossible to diagnose microorganisms expressing prolyl-peptidaseactivity, and in particular to separate yeast of the species Candidaalnicans from those of the species Candida tropicalis and Candidaglabrata.

EXAMPLE 13 Detection of Microorganisms Expressing Alanyl-PeptidaseActivity 13.1 Preparation of the Detection Media

The detection media were prepared by mixing 3.6 g of biogelytone(bioMérieux), 2.16 g of meat extract (bioMérieux), 1.26 g of maltextract (bioMérieux), 5.76 g of NaCl (Merck) and 10.8 g of agar(bioMérieux).

720 ml of osmosed water were added to the powder, and the mixture wasthen melted in a waterbath at 50° C. The product was separated into twoflasks, each of 360 ml. Autoclaving was carried out at 121° C. for 15min and the product was cooled to 100° C. in a waterbath.

L-Alanyl-7-amino-4-methylcoumarin (Ala-AMC, Bachem) was then added tothe first flask as a control, at the final concentration of 50 mg/l, andL-alanyl-9-aminobenzo[a]phenoxazin-5-one (Ala-ABP, substrate of theinvention obtained in Example 2, in which the amino acid is L-alanine),was then added to the second flask at the final concentration of 25mg/l.

Seeding of the Microorganism Strains

Twenty-five microorgansims strains derived from the Applicant'scollection, suspended in physiological saline, were seeded in colonieson each of the media. The dishes were incubated at 37° C. for 48 hours.The colonies formed were examined visually after incubation for 48hours. The size of the colonies, their coloration and also the intensityof this coloration were noted.

13.3 Results

The results are expressed in Table 4 hereinafter according to the rulesand the nomenclature stated in paragraph 11.3.

Table 4: Alanyl-peptidase Activity of Microorganisms with the SubstratesAla-AMC of the Prior Art and Ala-ABP of the Invention

Ala-AMC Ala-ABP T^(a) C^(b) I^(c) Co^(d) C^(b) I^(c) Co^(d)Acinetobacter baumanii 24 1.5 3 B 1.7 3 R 48 1.5 3 B 1.7 3 R Salmonellatyphimurium 24 1.3 3 B 1 0.5 R 48 1.5 3 B 1.5 1 R Proteus mirabilis 240.7 3 B 1.7 0.5 R 48 0.7 3 B 1.7 1.5 R Serratia liquefaciens 24 0.7 3 B0.7 3 R 48 1.3 3 B 1.3 3 R Serratia marcescens 24 0.5 3 B 0.5 2 R 48 0.73 B 1 3 R Hafnia alvei 24 0.7 3 B 0.5 0.5 R 48 0.7 3 B 0.5 1.5 REdwardsiella tarda 24 0.4 3 B 0.5 1 R 48 0.5 3 B 0.7 3 R Klebsiellapneumoniae 24 1.2 3 B 1 3 R 48 1.8 3 B 1.5 3 R Escherichia coli 24 1.7 3B 1.7 1 R 48 1.7 3 B 1.8 1 R Pseudomonas aeruginosa 24 0.3 0.3 1.7 R 480.5 2 B 0.5 3 R Enterobacter cloacae 24 0.5 3 B 0.7 1.5 R 48 0.8 3 B 0.72 R Streptococcus pyogenes 24 0.1 0.1 48 0.2 3 B 0.2 Enterococcusfaecium 24 0.2 0.2 48 0.3 0.25 B 0.3 Streptococcus agalactiae 24 0.1 0.148 0.2 0.2 Enterococcus faecalis 24 0.3 0.3 48 0.4 0.3 Staphylococcusepidermidis 24 0.3 0.1 48 0.4 0.1 Staphylococcus saprophyticus 24 0.30.1 48 0.4 0.3 Staphylococcus aureus 24 0.5 0.1 48 0.7 0.3 Bacillussubtilis 24 1.7 0.1 48 1.8 0.5 Corynebacterium 24 0 0Pseudodiphteriticum 48 0.4 2 B 0 Listeria innocua 24 0.2 0.2 48 0.3 0.3Saccharomyces cerevisiae 24 0.1 0 48 0.2 0.2 Candida krusei 24 0.7 0.548 1.5 0.25 1 Candida glabrata 24 0.2 0.1 48 0.3 0.3 Candida albicans 240.3 0.3 48 0.5 2 B 0.5 2 R

The results indicated in the table above demonstrate that thechromogenic enzymatic substrates of the invention clearly make itpossible to diagnose microorganisms expressing alanyl-peptidaseactivity, and in particular to separate Gram-positive bacteria (notexpressing the activity) from Gram-negative bacteria (expressing theactivity).

EXAMPLE 14 Detection of Microorganisms Expressing β-Alanine PeptidaseActivity 14.1 Preparation of the Detection Media

300 ml of Columbia medium were melted in a waterbath at 100° C. andautoclaved at 121° C. for 15 min. The product was then cooled to 50° C.in a waterbath.

This medium was separated into 3 flasks of 100 ml, and then, added at50° C., as substrates, were those of Examples 4 and 6 for which theamino acid is β-alanine (respectively,β-alanine-9-amino-6-carbethoxybenzo[a]phenoxazin-5-one or β-Ala-ACAP andβ-alanine-9-amino-6-chlorobenzo[a]phenoxazin-5-one or β-Ala-ACHP),solubilized in DMSO, according to the concentrations indicated in Table5 below: TABLE 5 Medium 1 Medium 2 Medium 3 β-Ala-ACAP 60 mg/lβ-Ala-ACHP 60 mg/l

The medium was then poured into Petri dishes.

14.2 Seeding of the Microorganism Strains

Nine microorganism strains derived from the Applicant's collection,suspended in physiological saline, were seeded onto the media preparedin point 14.1 above according to a multipoint inoculation in aproportion of 100 000 cfu/spot. The dishes were incubated at 37° C. for48 hours. The colonies formed (one colony per strain) were examinedvisually after incubation for 24 and 48 hours.

14.3 Results

The results are expressed in Table 6 hereinafter according to the rulesand the nomenclature stated in paragraph 11.3 and where RP signifiespale pink. TABLE 6 Medium 1 Medium 2 Medium 3 T^(a) C^(b) I^(c) Co^(d)C^(b) I^(c) Co^(d) C^(b) I^(c) Co^(d) Pseudomonas 24 3 3 1 R 3 2 Raeruginosa 1 48 3 3 3 R 3 2 RP Pseudomonas 24 3 3 2 R 2 2 R aeruginosa 248 3 3 2 R 3 2 RP Pseudomonas 24 1 1 2 R 1 2 R aeruginosa 3 48 2 2 3 R 22 RP Burkholderia 24 3 2 2 cepacia 1 48 3 2 2 Burkholderia 24 3 2 2cepacia 2 48 3 2 2 Escherichia coli 24 3 2 2 (NCTC 10418) 48 3 3 3Enterobacter 24 3 3 2 cloacae 48 3 3 3 (NCTC 11936) Enterococcus 24 3 21 faecalis 48 3 2 1 (NCTC 775) Candida albicans 24 3 2 2 (NCTC) 48 3 2 2

The results indicated in the table above demonstrate that thechromogenic enzymatic substrates of the invention clearly make itpossible to diagnose β-alanine peptidase activity specific for P.aeruginosa strains, all the other strains being negative for thisenzymatic activity revealed by these substrates.

EXAMPLE 15 Detection of Microorganisms Expressing Alanine PeptidaseActivity Using Substrates for A is at Least Two Amino Acids 15.1Preparation of the Detection Media

1000 ml of Columbia medium was melted in a waterbath at 100° C. andautoclaved at 121° C. for 15 min. It was then cooled to 50° C. in awaterbath.

This medium was separated into 5 flasks of 200 ml, and then, added at50° C., as substrates, was that of Example 2 for which A is:

-   -   medium 1: L-alanine (L-alanine-9-aminobenzo[a]phenoxazin-5-one        or A-ABP),    -   medium 2: L-alanine-L-alanine        (L-alanine-L-alanine-9-aminobenzo-[a]phenoxazin-5-one or        AA-ABP),    -   medium 3: L-alanine-L-alanine-L-alanine        (L-alanine-L-alanine-L-alanine-9-aminobenzo[a]phenoxazin-5-one        or AAA-ABP),    -   medium 4: L-alanine-glycine        (L-alanine-glycine-9-aminobenzo[a]phenoxazin-5-one or AG-ABP),        and    -   medium 5: glycine-L-alanine        (glycine-L-alanine-9-aminobenzo[a]phenoxazin-5-one or GA-ABP),        each substrate being solubilized in DMSO and used at a final        concentration of 50 mg/l.

The medium was then poured into Petri dishes.

15.2 Seeding of the Microorganism Strains

Nine microorganism strains derived from the Applicant's collection,suspended in physiological saline, were seeded in colonies on the mediaprepared in point 15.1 above according to a multipoint inoculation in aproportion of 15 000 cfu/spot. The dishes were incubated at 37° C. for48 hours. The colonies formed were examined visually after incubationfor 24 and 48 hours.

15.3 Results

The results are expressed in Tables 7 and 8 hereinafter according to therules and the nomenclature stated in paragraph 11.3 and where R=pink,RP=pale pink and I=colorless. TABLE 7 Medium 1 Medium2 Medium 3 T^(a)C^(b) Co^(b) C^(b) Co^(b) C^(b) Co^(b) Listeria monocytogenes 24 2 I 2 R2 R (NCTC 11994) 48 2 R 2 R 2 R Micrococcus luteus 24 0 0 1 R (NCTC 611)48 0 0 3 R Staphylococcus aureus 24 0 3 I 3 I (NCTC 6571) 48 2 I 2 I 3 IStaphylococcus epidermis 24 0 2 I 2 I (NCTC 11047) 48 0 2 I 2 IAcinetobacter baumannii 24 3 R 3 R 3 R (ATCC 19606) 48 3 R 3 R 3 REnterobacter clocae 24 3 R 3 R 3 R (NCTC 11936) 48 3 R 3 R 3 REscherichia coli 24 3 R 3 R 3 R (NCTC 10418) 48 3 R 3 R 3 R Pseudomonasaerigunosa 24 3 R 3 R 3 R (NCTC 10038) 48 3 R 3 R 3 R

TABLE 8 Medium 1 Medium 4 Medium 5 T^(a) C^(b) Co^(b) C^(b) Co^(b) C^(b)Co^(b) Listeria monocytogenes 24 2 I 2 I 2 I (NCTC 11994) 48 2 R 3 I 2 RMicrococcus luteus 24 0 1 I 0 (NCTC 611) 48 0 3 R 0 Staphylococcusaureus 24 0 3 I 3 I (NCTC 6571) 48 2 I 3 I 3 I Staphylococcus epidermis24 0 3 I 1 I (NCTC 11047) 48 0 3 I 2 I Acinetobacter baumannii 24 3 R 3RP 3 R (ATCC 19606) 48 3 R 3 R 3 R Enterobacter clocae 24 3 R 3 R 3 R(NCTC 11936) 48 3 R 3 R 3 R Escherichia coli 24 3 R 3 R 3 R (NCTC 10418)48 3 R 3 R 3 R Pseudomonas aerigunosa 24 3 R 3 RP 3 RP (NCTC 10038) 48 3R 3 R 3 R

The results indicated in Tables 7 and 8 above demonstrate that, whateverthe length of the amino acid chain of the chromogenic enzymaticsubstrates of the invention, the latter make it possible to detect theenzymatic expression of microorganism strains having alanine peptidaseactivity. In addition, due to the nature and the number of amino acids,it is possible to vary both the specificity and the sensitivity or thetoxicity of the substrates for the diagnosis of said strains accordingto the strain under consideration.

EXAMPLE 16 Detection of Microorganisms by Combining a Substrate of theInvention and a Substrate of the Prior Art 16.1 Preparation of theDetection Medium

3.3 g of yeast extract, 2.75 g of Biocase, 2.475 g of malt extract,0.165 g of glucose and 7.7 g of agar were mixed and 550 ml of osmosedwater were added.

The mixture was melted in a waterbath at 100° C. and autoclaved at 121°C. for 15 min. It was then cooled to 50° C. in a waterbath.

The substrate of the invention prepared in Example 2, in which the aminoacid is proline, was added in a proportion of 0.05 g/l and5-bromo-4-chloro-3-indoxyl-β-D-glucoside was added, as other substrateof the prior art, in a proportion of 0.05 g/l.

The medium was then distributed into Petri dishes.

16.2 Seeding of the Microorganism Strains

Twelve microorganism strains derived from the Applicant's collection,suspended in physiological saline, were seeded in colonies on the mediumprepared in point 16.1 above. The dishes were incubated at 37° C. for 48hours. The colonies formed were examined visually after incubation for24 and 48 hours.

16.3 Results

The results, indicated in Table 9 below, are expressed as growth withthe size being indicated in mm, as activity and as color, Tarepresenting the incubation time, R=pink, O=orange, V=green,T=turquoise, M=brown, GO=orangey-gray and GV=grayish-green. TABLE 9Strain T^(a) Growth Activity Color Escherichia coli 24 h 3 2 R 48 h 3 3O Serratia marcescens 24 h 1.5 2 O 48 h 1.7 2 O Serratia liquefaciens 24h 1.5 2 GV 48 h 1.5 2 M Klebsellia pneumoniae 24 h 2 3 GV 48 h 3 3 GOMorganella morganii 24 h 2 48 h 2 2 O Acinetobacter baumanii 24 h 3 2 R48 h 3 3 R Hafnia alvei 24 h 2 0.5 R 48 h 2.5 3 R Edwardsiella tarda 24h 1.5 0 O 48 h 1.7 1.5 R Pseudomonas aeruginosa 24 h 1 2 O 48 h 1.5 3 MListeria innocua 24 h 0.5 3 T 48 h 0.5 3 T Staphylococcus sciuri 24 h0.5 3 T 48 h 0.5 3 T Enterococcus faecalis 24 h 1.2 3 T 48 h 1.2 3 TCandida albicans 24 h 1.2 2 R 48 h 1.5 3 R

The results indicated in Table 9 above show that a colorationcharacteristic of the enzymatic activities exhibited by the strains isclearly observed, as follows:

-   -   a pink to orange coloration in the case of the strains having        only the proline peptidase activity due to the hydrolysis of the        substrate of the invention (Escherichia coli, Morganella        morganii, Acinetobacter baumanii, Hafnia alvei, Edwardsiella        tarda and Candida albicans),    -   a turquoise coloration in the case of the strains having only        the β-glucosidase activity due to the hydrolysis of the        substrate of the prior art (Staphylococcus sciuri, Enterococcus        faecalis) and    -   a green to brown coloration, originating from the mixture of the        2 colorations pink/orange and turquoise, for the strains having        the two enzymatic activities (Serratia marcescens, Serratia        liquefaciens, Klebsellia pneumoniae, Pseudomonas aeruginosa and        Listeria innocua).

Consequently, it is possible to detect several different enzymaticactivities specific for each strain according to their metabolism.

1. A chromogenic enzymatic substrate, characterized in that itcorresponds to formula (D) below:

in which R₁ and R₂ form, with the phenyl ring to which they areattached, a naphthalene ring of formula:

or an optionally substituted coumarin ring of formula:

or else R₁ and R₂ each independently represent a hydrogen atom, a C₁-C₆alkyl group, a halogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an—SO₃H group or a sulfonamide group, R₃ and R₄ form, with the phenyl ringto which they are attached, an optionally substituted naphthalene ringof formula:

or else R₃ and R₄ each independently represent a hydrogen atom, ahalogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, an —SO₃H group or asulfonamide group, it being understood that: (i) at least one amongR₁/R₂ and R₃/R₄ forms, with the phenyl ring to which it is attached, anoptionally substituted naphthalene or coumarin ring as defined above,and (ii) when R₁ and R₂ form, with the phenyl ring to which they areattached, an optionally substituted coumarin ring, R₃ and R₄ do notform, with the phenyl ring to which they are attached, an optionallysubstituted naphthalene ring, R₅ and R₆ each independently represent ahydrogen atom, a halogen atom, a —C(O)OR′ group, a C(O)NR′R″ group, or aC₁-C₆ alkyl group, it being understood that R₆ represents a halogen atomwhen R₁/R₂ and R₃/R₄ each form, with the phenyl ring to which they areattached, a naphthalene ring, R₇ and R₈ each independently represent ahydrogen atom, a C₁-C₆ alkyl group, an aralkyl group, an aryl group, acarboxyalkyl group, a carboxyl group or a sulfonic acid group, or elseR₇ and R₈, together with the two carbon atoms to which they areattached, form a C₄-C₆ ring, R₉ represents a hydrogen atom, a bromineatom, a chlorine atom, a benzoyl group, a —CO₂H group or an —SO₃H group,it being understood that, when R₉ is different from a hydrogen atom,then R₅ is a hydrogen atom, R′ represents a hydrogen atom or a C₁-C₆alkyl group, R″ represents a hydrogen atom or a C₁-C₆ alkyl group, orelse R′ and R″, together with the nitrogen atom to which they areattached, form a heterocyclic ring containing one or more hetero atoms,A represents at least one amino acid, and X represents a blocking agentor nothing.
 2. The chromogenic enzymatic substrate as claimed in claim1, characterized in that there is one and only one among R₁/R₂ and R₃/R₄forms, with the phenyl ring to which it is attached, an optionallysubstituted naphthalene or coumarin ring.
 3. The chromogenic enzymaticsubstrate as claimed in claim 2, characterized in that it corresponds toformula (Ia) below:


4. The chromogenic enzymatic substrate as claimed in claim 3,characterized in that R₅ represents a hydrogen atom, R₆ represents ahydrogen atom or a halogen atom, A is an amino acid chosen from leucine,proline and alanine, and X is the t-butoxycarbonyl blocking agent ornothing.
 5. The chromogenic enzymatic substrate as claimed in claim 2,characterized in that it corresponds to formula (Ib) below:

in which R₅, R₇, R₈, A and X are as defined in claim
 1. 6. Thechromogenic enzymatic substrate as claimed in claim 5, characterized inthat R₅ is a hydrogen atom, R₇ and R₈ each independently represent ahydrogen atom, a C₁-C₆ alkyl group, an aralkyl group, an aryl group or acarboxyalkyl group, or else R₇ and R₈, together with the two carbonatoms to which they are attached, form a C₄C₆ ring, A is an amino acidchosen from leucine, proline and alanine 1, and X is thet-butoxycarbonyl blocking agent or nothing.
 7. The chromogenic enzymaticsubstrate as claimed in claim 2, characterized in that it corresponds toformula (Ic) below:


8. The chromogenic enzymatic substrate as claimed in claim 7,characterized in that R₅, R₆ and R₉ each represent a hydrogen atom, A isan amino acid chosen from leucine, proline and alanine and X is thet-butoxycarbonyl blocking agent or nothing.
 9. The chromogenic enzymaticsubstrate as claimed in claim 1, characterized in that it corresponds toformula (Id) below:


10. A reaction medium that uses at least one chromogenic enzymaticsubstrate as claimed in claim 1, alone or in combination with at leastone other enzymatic substrate specific for an enzymatic activitydifferent from that detected by the substrate according to theinvention.
 11. The medium as claimed in claim 10, characterized in thatit is a culture medium.
 12. The medium as claimed in claim 10,characterized in that it is in gelled form.
 13. A chromogenic enzymaticsubstrate as defined in claim 1, for at least one of the detectionand/or, identification and quantification of microorganisms expressingat least one peptidase activity.
 14. A method for the detection and/oridentification and/or quantification of microorganisms expressing atleast one peptidase activity, characterized in that it consists in:providing a reaction medium, as defined in claim 10, seeding the mediumwith a biological sample to be tested, leaving to incubate, andrevealing the presence of at least one peptidase activity alone or incombination with at least one other enzymatic activity different fromthis same peptidase activity.
 15. A method for differentiating, amongbacteria, between those belonging to Gram-positive microbes and thosebelonging to Gram-negative microbes, characterized in that it consistsin: providing a reaction medium, as defined in claim 10 and in which thesubstituent A of the chromogenic substrate is alanine, seeding themedium with a biological sample to be tested, leaving to incubate, andrevealing the presence of at least one coloration synonymous with thepresence of a Gram-negative microbe or Gram-negative microbes.
 16. Amethod for differentiating the yeast of the species Candida albicansfrom those of the species Candida tropicalis and Candida glabrata,characterized in that it consists in: providing a reaction medium, asdefined in claim 10 and in which the substituent A of the chromogenicsubstrate is proline, seeding the medium with a biological sample to betested, leaving to incubate, and revealing the presence of at least onecoloration synonymous with the presence of yeast of the species Candidaalbicans.
 17. A reaction medium as defined in claim 10 for at least oneof detection, identification and quantification of microorganismsexpressing at least one peptidase activity.